The mechanism(s) underlying the initiation and progression of autoimmune diseases are not well understood. Epidemiological evidence strongly suggests a viral etiology for a number of autoimmune diseases including multiple sclerosis (MS), a human CD4+ T cell-mediated demyelinating disease associated with antimyelin responses. Postulated mechanisms by which virus infections may initiate or exacerbate autoimmune disease include: (1) virus-induced bystander activation of autoreactive T cells; (2) epitope spreading (de novo activation of autoreactive T cells by sequestered antigens released secondary to self tissue destruction); and (3) molecular mimicry (activation of autoreactive T cells secondarily by viral epitopes shared or cross-reactive with self antigens). Our studies in the Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease model of MS have shown that CNS demyelination in SJL mice infected with the wildtype BeAn strain of TMEV is initiated by virus-specific CD4+ Th1 cells targeting virus persisting in CNS macrophages/microglia. Autoreactivity to the immunodominant myelin proteolipid protein epitope, PLP139-151, arises 6-7 weeks post infection via epitope spreading and plays a major pathologic role in chronic progression. During the past grant period, we developed and extensively characterized a virus-induced molecular mimicry model of CNS demyelination by infecting mice with a non-pathogenic variant of TMEV engineered to express PLP139-151 or a molecular mimic (residues 574-586 of H. influenzae protease IV (sppA) - HI574-586) which shares only 6/13 amino acid residues with the encephalitogenic self epitope. SJL mice infected with HI574-TMEV develop a rapid-onset clinical demyelinating disease mediated by autoreactive PLP139-151-specific T cells. In the current proposal, we will further test the hypothesis that induction of CNS autoimmunity by virus-induced molecular mimicry depends both on expression of a mimic epitope and on the ability of the pathogen to stimulate critical innate immune signals required to activate autoreactive T cells. Aim 1 will further test the molecular mimicry hypothesis by assessing the efficiency of autoreactive T cell activation (or regulation), T cell repertoire composition, and induction of initial clinical disease and relapses following infection with TMEV encoding various molecular mimics of PLP139-151 naturally expressed by MHV, E. coli, C. albicans, or S. typhimurium in addition to TMEV expressing regulatory PLP139-151 altered peptide ligands. The nature of the virus-induced innate immune signals required to initiate mimicry-induced CNS disease and the ability of infection with non-neurotropic/non-persisting viruses to initiate CNS disease will be assessed. Aim 2, relevant to human MS, will assess activation/regulation of autoreactive T cells and clinical disease induced by infection of humanized HLA-DR2b/MBP85-99-specfic TCR double transgenic mice with TMEV encoding various molecular mimics of the MS-associated MBP85-99 epitope naturally expressed by common bacterial pathogens. Lastly, Aim 3 will advance our studies to investigate the mechanisms of naturally occurring molecular mimicry. The ability of wildtype vs. a spp/A-deficient strain (lacking the HI574-586 mimic) of H. influenzae to activate PLP139-151-specific T cell responses and to induce CNS disease will be tested in both conventional and 5B6 PLP139-151-specific SJL TCR transgenic mice. These studies will enhance our understanding of the mechanisms by which natural infections may initiate/exacerbate MS and other autoimmune diseases by defining the activation requirements and mechanisms by which autoreactive T cells are activated by viral infection. [unreadable] [unreadable] [unreadable]